One of the first steps of DNA replication is the loading of the replication proteins onto the DNA that is to be replicated. The T4 gene 59 protein plays a key role in this process and greatly stimulates the loading of T4 gene 41 helicase at the site of replication, leading to the formation of the complete protein complex necessary for efficient replication of the T4 genome. Similar events are thought to happen during the initiation of DNA replication in all organisms, so the insight gained through studying the biochemistry of gene 59 loading protein may help to illuminate general mechanisms involved in DNA replication. Previously, our biochemical analyses and a crystal structure of the gene 59 protein obtained in collaboration with Dr. Tim Mueser (University of Toledo) have suggested that this protein binds to the fork formed at the junction between replicated and unreplicated DNA and delivers 41 helicase to this site. We have been using mutational analyses to define the regions of the 59 protein that are required for its interaction with the replication fork and with the T4 helicase, polymerase, primase, and single-stranded DNA binding protein. We have constructed plasmids encoding 59 protein with mutations of residues that were highly conserved in T4 related phage, or with mutations of residues that appeared interesting from the structural analysis. We have expressed, purified, and begun to characterize 16 of these mutants both in vitro and in vivo. In vitro characterization by gel mobility shift assays has revealed that several mutants are defective in binding fork DNA substrates that mimic the start sites for replication in vivo. Our analyses suggest that the N-terminal domain of 59 protein recognizes the distorted fork DNA structure, bends the duplex DNA upon binding, and holds the fork open to allow loading of 41 helicase and assembly of the T4 replisome.